Method and devices for measuring distances between object structures

ABSTRACT

The invention concerns a method and devices for far field microscopy and flow fluorometry for geometric distance measurements between object structures, i.e. measurement structures, marked with fluorochromes, whereby the distances can be smaller than the half-intensity width of the principle maximum of the point spread function. In this method, the measurement structures are marked with fluorescent dyes with different or identical spectral signatures, according to their distances. Calibration targets with defined dimensions and arrays are marked with the same fluorescent dyes. Calibration targets and measurement structures are prepared separately or together on an object support and investigated microscopically or flow fluorometrically. In each case, two defined calibration targets with different spectral signatures are measured in consideration of the wavelength dependent imaging and localisation behaviour of the optical system used, the measurement values thus obtained are compared against the previously known real distance values and the difference is used as the calibration value for correcting the shift in the direction of the measurement structures caused by the optical system. In the case of the devices, these are calibration targets and an axial tomograph for undertaking the method described above.

FIELD OF THE INVENTION

The invention relates to a method and devices for far field microscopyand flow fluorometry for geometric distance measurements between objectstructures marked with fluorochromes, wherein the distances may besmaller than the half-intensity width of the principle maximum (=fullwidth at half maximum of the intensity peak=FWHM) of the actual pointspread function.

BACKGROUND INFORMATION

By employing highly specific markers, such e.g. DNA probes or proteinprobes, it is possible to mark practically any small (sub-)structures inbiological (micro-)objects, in particular in cells, cell nuclei, cellorgans or chromosomes (hereinafter also described as objects forabbreviation). Such markers may specifically represent structures indimensions from several μm (10−6 m) to a few tens of nm (10−9 m). Inthese markers normally reporter molecules are integrated, which have ahigh affinity with complex compounds, to which fluorochromes, but alsocolloidal microparticles (e.g. gold) are attached. Suchfluorochromes/complexes can also be integrated directly into themarkers. The available colour emission spectra of fluorochromes stretchfrom deep blue through green and red to the infrared range of thespectrum. Equally, fluorochromes can be used that do not differentiatein terms of excitation and/or fluorescence emission in their spectrum,but in which the life time of their fluorescence emission is used as aparameter for differentiation.

The latter have the advantage that focal shifts depending on thewavelength do not arise. Fluorochromes can also have a differentemission spectrum and thus possess different spectral signatures, andyet be stimulated by the same photon energy, e.g. by means of multiplephoton processes. It is also possible in this case to avoidwavelength-dependent focal shifts in the excitation betweenfluorochromes with different spectral signatures.

The above-named fluorochromes bound to specific (sub-) structures inbiological micro-objects are referred to hereinafter as fluorescencemarkers. Fluorescence will be used below to encompass every photoninteraction in which differences arise between a material's stimulationspectrum and its emission spectrum that cannot be attributed tomonochromatic absorption or dispersion. This also includes in particularmultiple photon interactions in which the stimulation wavelengths may begreater than the emission wavelengths. Furthermore, the termfluorescence is also used here for the closely related phenomenon ofluminescence, in particular phosphorescence. This includes in particularlonger and medium term fluorescence life time, e.g. fluorescence lifetime in the range of up to several or many msec (milliseconds). Theclosely related processes of luminescence, phosphorescence andfluorescence will be treated herein as equally relevant to theinvention. If the excitation spectrum and/or the emission spectrumand/or the fluorescence life time of two fluorescence markers agree,they have the same spectral signature based on the parameter inquestion. If they differ in one or more parameters relevant to themeasurement, they have different spectral signatures.

A series of light microscopic measuring methods is used for detectingthe fluorescence markers in extended biological objects and for thequantitative localisation relative to defined object points/objectstructures (distance and angle measurements). This is primarily a caseof (a) epifluorescence microscopy, (b) confocal laser scanningmicroscopy, (c) laser scanning flow fluorometry, (d) the far fieldmicroscopy process of “point-spread-function-engineering” and (e)standing wave field microscopy.

a) In the case of epifluorescence microscopy with a classical upright orinverse epifluorescence microscope, the biological object is illuminatedby the same lens through which it is detected. The excitation light andthe fluorescence emitted are discriminated by appropriate opticalfilters and conducted into different beam paths. The obtainableresolution, i.e. the smallest distance still measurable between twopoint-shaped object structures that are marked with fluorochromes withthe same spectral signature, is given either by the Abbe criterion (=themaximum 0. order of the diffraction pattern of a point object islocalised in the 1st minimum of the diffraction pattern of a secondpoint object) or by the half-intensity width of the principle maximum ofthe actual point spread function. This depends on the wavelength, on thenumerical aperture of the lens used and on the local refractive indicesof the objects, of the embedding medium, of any cover slip used and ofany immersion fluid applied. (In the case of a higher numericalaperture, its dimension may be smaller than the wavelength of the lightused for stimulation).

b) In the case of confocal laser scanning microscopy, unlikeepifluorescence microscopy, a laser is focused in the lens and thefluorescence is detected confocally. In order to create athree-dimensional image, the object is scanned in all three directions(x, y, z) with the focus point. As in the case of epifluorescencemicroscopy, the obtainable resolution is given by the half-intensitywidth of the principle maximum (FWHM)of the actual point spread functionand depends on the wavelengths, on the numerical aperture of the lensused and on the local refractive indices of the objects, of theembedding medium, of any cover slip used and of any immersion fluidapplied.

c) In the case of laser scanning flow fluorometry, the objects areconducted for example individually through an appropriate lightdistribution of the focus by a carrier fluid beam that is free orsituated or in an optical cuvette (while in the case of epifluorescencemicroscopy and confocal laser scanning microscopy, the objects arepredisposed in a fixed position on object carriers, i.e. object slides,capillaries, chambers, fluids etc.). The light distribution is normallyslit-shaped, i.e. the object is scanned with reference to an axis. Theobtainable resolution is determined by the width of the focus of thelaser beam used and/or suitably selected detection scans, wherein thevariability in the object trajectory (=laminar, usually central “fluidfilaments” that carry the object) allows for the focal depth and thusalso the minimal focal width, regardless of the carrier medium andmethod. The advantage of flow fluorometric methods is usually found inthe relatively higher detection rate, compared to epifluorescencemicroscopy and confocal laser scanning microscopy, which can reachseveral thousand objects per second. The focal width complies with thefull half-intensity width of the principle maximum of the actual pointspread function of the slit-scan optics in the conditions used.

d) In the case of the far field microscopy technique of“point-spread-function-engineering”, the point spread function isreduced in width optically. This may be achieved by means of coherentlyoverlapping two or more point spread functions (e.g. 4Pi microscopy) orby means of extinguishing the fluorescence of fluorochromes that aresituated in the peripheral area of the central point spread functionmaximum in question (e.g. STED microscopy, ground depletion microscopy).As the resolution of a microscope is given by the full half-intensitywidth of the principle maximum of the actual point spread function, thehalf-intensity width is thus reduced and the resolution improved.

e) In the case of standing wave field microscopy according to U.S. Pat.No. 4,621,911, luminescent preparations are illuminated with a standingwave field in an optical microscope (standing wave field fluorescentmicroscopy, SWFM). The preparations are set in a zone of equidistantwave fronts and stimulated to fluorescence or phosphorescence. The spacebetween the wave fronts and their phases can be varied to generatepatterns. The three-dimensional distribution of fluorescent orluminescent object points can be reconstructed from individual opticalsections by means of computer image processing.

The planar wave fronts are generated by coherently overlapping two laserbeams at a defined angle to the optical axis of the microscope system,whereby the angle determines the distance between the wave fronts—withgiven wavelengths and refraction indices. Instead of two intersectinglaser beams, the standing wave field can also be generated by making alaser beam interfere with itself at a certain angle after suitablereflection. In microscope construction in these cases, the wave frontsare set perpendicularly to the optical axis of the detecting lens. Thefluorescence or luminescence is either spectrally discriminated by meansof appropriate optical filters and conducted into different beam paths,as in the epifluorescence microscope, or detected confocally. As in thecase of epifluorescence microscopy and confocal laser scanningmicroscopy, the obtainable resolution is given by the fullhalf-intensity width of the principle maximum of the actual point spreadfunction and depends on the given wavelength, on the numerical apertureof the lens used and on the local refractive indices of the objects, ofthe embedding medium, of any cover slip used and of any immersion fluidapplied.

Laterally, the system has a resolution akin to a conventionalepifluorescence microscope or a confocal laser scanning microscope;axially, on the other hand, a depth discrimination and thus aconsiderably better resolution is achieved.

DISADVANTAGES OF THE STATE OF THE ART

1) As the actual point spread functions are strongly influenced by thelocal refraction index and absorption in the object, in the object'sembedding medium and in the immersion (including any cover slipspresent), the measurement of distances between object structures dependson the actual point spread function-i.e. the one given locally in themarked object point. This generally differs clearly from calculatedpoint spread functions of the microscope used. The technically optimisedmarginal conditions of measured point spread functions also generallydiffer from the actual point spread functions obtainable in biologicalobjects under practical routine laboratory conditions. As these actualpoint spread functions are mostly not available, distance measurementsfor calibrating purposes are usually made by referring to ideal,calculated results or to calibration measurements that are made understandard conditions, such as e.g. reflection methods. Both methods aredetrimental to accuracy in three-dimensional distance measurement inbiological micro-objects. As a result, there is considerable uncertaintyin the determination of the real spatial distance between the objectstructures; in the case of biological objects, quantitative sizeestimates contain uncertainties of up to several micrometers. There isonly a limited possibility to correct this error with the methods usedhitherto, i.e. only under standard conditions, whose actualachievement/compliance in the biological object cannot be controlled orguaranteed accurately, however.

Two object structures with the same spectral signature can then only beseparated if the distance between them is at least a half-intensitywidth of the principle maximum of the actual point spread function.

2) All the ar field methods described above suffer from the problem thatthe half width of the principle maximum of the point spread function andthus the limits of resolution depend on the relative position in space.Thus, in the case of epifluorescence microscopy or confocal laserscanning microscopy, for example, the point spread function is narrowerlaterally (perpendicular to the optical axis) than axially (in thedirection of the optical axis). In the case of static microscopymethods, this disadvantage can be overcome with the aid of so-calledmicro-axial tomography. In this method, the (biological) objects are setin capillaries or on glass fibres and rotated around an axis underdefined conditions in the microscope, which is normally perpendicular tothe microscope's optical axis. In this case, distance measurements aremade in the direction with the narrowest half-intensity width of theactual point spread function. This method can hardly be applied,however, in the case of flow fluorometry.

3) In the case of “mono-dimensional” standing wave field microscopy(SWFM), the periodical wave field in the case of epifluorescentdetection in connection with optical sectioning leads to ambiguity inthe image of the object structures greater than λ/2 n (λ=wavelength ofthe excitation, n=actual refraction index). This ambiguity primarilymakes it difficult to make any effective use of the improvement inresolution achieved by means of the interference pattern.

4) According to the state of the art, high precision distancemeasurements with light microscopy far field methods can only be made asfar as the range of dimensions of a hundred nanometers. For measurementsin the range of distances and accuracy of the order of 10 nm, themethods of electron microscopy, scanning tunnel microscopy, atomic forcemicroscopy and biological and optical near-field microscopy are used.Nevertheless, these are surface-oriented and not volume-orientedmethods-unlike the optical far field method; in other words, they are inprinciple useful only for structural investigations and distancemeasurements on surfaces and in thin layers. In any event, informationabout the position of objects or object structures in three dimensionscan be obtained using mechanically prepared cut series and evaluatingmeasurements in individual image sections. Three-dimensionalmeasurements in intact or even vital biological micro-objects, such asthree-dimensional (conserved) cells, cell nuclei or cellular organs, arenot possible.

SUMMARY OF THE INVENTION

The task of the invention is to prepare a method for far field lightmicroscopy and a device for executing this method, with which it ispossible to make highly accurate distance measurements between objectstructures whose reciprocal distance is smaller than the resolutioncapacity of the far field microscope in question, i.e. that areseparated by less than the half-intensity width of the maximum of theactual point spread function, regardless of the position of the objectstructures in question in the three-dimensional space.

One solution to this task consists of preparing a method of the kinddescribed above that is a calibrating process for fluorescence far fieldmicroscopy and includes the following process steps:

Before, during or after the preparation of the object in question on orin an object carrier, in particular object slides, fibres/capillaries orfluids, the structures to be investigated or localised (measurementstructures) are labelled with fluorescent dyes with different and/oridentical spectral signatures, i.e. such structures (measurementstructures) to be localised as are located in each others' immediatevicinities, i.e. within the half-intensity width of the principlemaximum of their actual point spread function, are labelled withfluorescent dyes with different spectral signatures, while suchmeasurement structures as are located at distances greater than that ofthe half-intensity width of the principle maximum of the actual pointspread function are labelled with fluorescent dyes with different orequal spectral signatures. Two measurement structures to be localisedcan thus always be labelled with the same spectral signature, if forexample they can be identified clearly by means of their relativepositions or other criteria.

calibration targets of defined sizes and spatial arrays are labelledwith the same fluorescent dyes,

the fluorescent calibration targets are prepared, either together withthe objects or separately on or in an object carrier (object slides,fibres/capillaries, fluids or the like).

objects (of investigation) and calibration targets are investigatedmicroscopically or flow fluorometrically in corresponding conditions,simultaneously or one after another.

pairs of defined calibration targets with different spectral signaturesare measured in consideration of the wavelength-dependent imaging andlocalisation behaviour of the optical system in question (microscope orflow fluorometer); the values thus measured—the actual values—arecompared to the previously known real distance values—the nominal values(i.e. the nominal localisation calculated on the basis of geometry), andthe difference between the actual values and the nominal values, i.e.the calibration value, is used to correct the offset caused by theoptical system in the detection of different emission loci, inparticular in the measurement structures.

In other words, the distance measurement between the object (sub-)structures—hereinafter also described as measurement structures—marked(according to the distances between them) with different or identicalspectral signatures is made using the high precision localisation ofindependent (calibration) targets with a conforming spectral signaturewith a known size and spatial array, in consideration of thewavelength-dependent imaging and localisation behaviour of the opticalsystem in question, whereby the calibration measurement between the(calibration) targets and the measurement in the biological objectstakes place in the same system and marginal conditions. Thesecalibration targets have the same or a higher multi-spectrality as orthan the (object) structures to be measured. They can be arrayeddirectly in the biological objects or as a separate preparation on anobject carrier (object slides, fibres/capillaries, fluids or the like)or be part of an object carrier.

Two or more fluorescent measurement structures in intact,three-dimensional biological objects, whose distance and extension isless than the half-intensity width of the principle maximum of theiractual point spread function, can be discriminated on the basis of theirdifferent spectral signature (fluorescence absorption wavelengths and/orfluorescence emission wavelengths and/or fluorescence emission lifetime), i.e. their distances can be determined.

The distance measurement can be reduced to the localisation of theindividual measurement structures and can—now also in optical far fieldmicroscopy or flow fluorometry—by carried out with a considerably higheraccuracy than the half-intensity width of the maximum of the pointspread function. The localisation of the barycentre of the measurementstructures in question is adapted to the maximum intensity of theirfluorescence signals. In other words, the barycentre of the signal andthus the location of the measurement structure are determined from themeasured (diffraction limited) signal (=intensity curve) of afluorescence point (=fluorescence measurement structure)-inconsideration of the overall information from the minor and majormaxima. In the case of an error-free optical system and consequentlyideal symmetry of the measured intensity distribution (=course of theintensity curve), the barycentre of the intensity curve co-localiseswithin the localisation accuracy with the main maximum (=maximum 0.order of the diffraction pattern) of the measured intensitydistribution.

The method in accordance with the present invention enables optical farfield region microscopy or scanning flow fluorometry to be used tomeasure distances in biological micro-objects, whereby the distances tobe determined may be smaller than the half-intensity width of theprinciple maximum of the actual point spread function in the object. Asthe information content of the distance determination carried out inaccordance with the invention complies with a distance measurement madewith higher resolution, it is also possible to speak summarily in termsof resolution equivalent. The measurements of such small distances inbiological micro-objects is of great significance for example forscientific questions in biology and medicine, but also for certainaspects of clinical research and diagnostics of preparations.

Multispectral calibration allows in situ measurements to be made ofconcrete biological objects via the system's image behaviour. Iffluorescence life time is used as the only type of parameter and/or ifthe fluorochromes are excited with the same photon energy/ies, the insitu correction of the chromatic shift in the object plane is no longernecessary. For high resolution far field microscope types, such as e.g.the standing wave field microscope, and when fluorescence markersaccording to the invention are used, the invention enablesthree-dimensional geometric distance measurements to be made inbiological objects right down to molecular accuracy (i.e. resolutionequivalent better than 10 nm).

Unlike electron microscopy or optical or non-optical near fieldmicroscopy, the three-dimensional structure of the object to beinvestigated remains intact, as there is no need for mechanicalsectioning. 3D distance measurements with a range smaller than thehalf-intensity width of the maximum of the actual point spread functioncan thus be undertaken in three-dimensionally conserved micro-objects.In particular, the method makes it possible to undertakethree-dimensional distance measurements also in vital conditions of thebiological object. Compared to the methods of point spread functionengineering known to the state of the art, a considerable advantage inthe invention consists of the fact that already existing systems forquantitative fluorescence microscopy can also be used as a basis for theincrease according to the invention of the resolution equivalent.

Both one and two-dimensional scanning and also non-scanning electronicor opto-electronic detector systems are suitable for detecting thefluorescence emission.

One application of the method according to the invention is particularlyuseful for multispectral precision distance measurements in biologicalmicro-objects for the absolute and relative localisation and distancemeasurement of fluorescent measurement structures with any spectralsignature.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a mounting according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to achieve as sharp a point spread function as possible whenusing static microscope systems, it should be possible to turn thebiological object of the microscopic investigation axial-tomographically(micro-axial tomography). In this way, anisotropies in the 3D pointspread function can be overcome so that two object points at a time layin a plane with the given best point spread function. The biologicalobject is preferably fixed in or on a rotating carrier with a circular,quadrangular or polygonal cross-section or fixed in some other way.

The carrier consists of a material transparent to the light wavelengthsused, whose refraction index differs from the surrounding medium by 12percent at the most. It may be hollow or solid and its cross-sectionaldiameter should be less than or equal to 300 μm.

In one preferred embodiment of the method according to the invention,the carrier (with a triangular, rectangular or polygonal cross-section)for the axial tomographic investigations is rotated through the angleφ_(m)=360/3[∘], φ_(m)=360/4[∘], or φ_(m)=360/n [∘], where n is thenumber of the planar sides of the carrier. A distance measurementbetween the calibration targets and/or measurement structures isundertaken at one, several or each of these angles, in each case forone, two or several spectral signatures.

The following method steps are preferably undertaken to determine theactual and nominal values to compare them and define thecorrection/calibration value:

one or more calibration targets B at a distance greater than thehalf-intensity width of the principle maximum of the actual point spreadfunction from the barycentre of the N measurement structures is/aremarked with a random spectral signature;

the distances d_(ik) (i, k=1 . . . N, i≠k) of the barycentres of thespectrally separated diffraction figures of the N measurement structuresand the distances d_(iB) of the N measurements structures to thecalibration target B are measured, using automated image analysisprocesses;

for one measurement structure, the stretches d_(ik) and d_(iB) at themoment in the plane of the narrowest point spread function and all otherdistances are measured, while the object is axial-tomographicallyrotated each time through a defined angle φ_(m);

optical aberrations from the calibration measurements are corrected, anda cosine function A_(ik) cos (φ_(m)+Θ_(ik)) or A_(iB) cos (φ_(m)+Θ_(iB))with suitable phase dislocation is adapted on each occasion to thecorrected measured distances d_(ik) (φ_(m)) and d_(iB) (φ_(m));

the maxima A_(ik) and A_(iB) of the adaptation function of d_(ik) ord_(iB) are divided by the multiplication factor and determined as aEuclidean distance D_(ik) or D_(iB) between the N measurement structuresor between the measurement structures and the point of reference B.

In order to determine the maxima, the relative minima of the distanceZ_(ik), Z_(iB) in the plane orthogonal to the plane of the d_(ik),d_(iB) should preferably be involved and analogically evaluated.

According to the invention, all the co-ordinates of the N measurementstructures and their co-ordinates with relation to the point ofreference B, i.e. the positions x_(i), y_(i), z_(i) and x_(k), y_(k),z_(k), as well as the distances x_(k)−x_(i), y_(k)−y_(i), z_(k)−z_(i)and x_(B)−x_(i), y_(B)−y_(i), z_(B)−z_(i), are identified on the basisof the microscopically measured 3D distances D_(ik) or D_(iB),preferably using the following system of equations:D_(ik)² = (x_(k) − x_(i))² + (y_(k) − y_(i))² + (z_(k) − z_(i))²D_(iB)² = (x_(B) − x_(i))² + (y_(B) − y_(i))² + (z_(B) − z_(i))²D_(kb)² = (x_(B) − x_(k))² + (y_(B) − y_(k))² + (z_(B) − z_(k))²

To guarantee the measurement results determined, the method describedabove should be carried out for several calibration targets B and thesame N measurement structures.

The co-ordinates and distances of the N measurement structures can bedetermined using the barycentres resulting from the barycentre centeringof the measurements to all the points of reference.

In particular for graphic representations, the positions determinedx_(i), y_(i), z_(i) and x_(B), y_(B), z_(B) are preferably convolvedwith a point spread function with a half-intensity width with theresolution equivalent achieved in each case.

For the purposes of fluorochrome labelling of the measurement structuresand calibration targets, such fluorochromes are preferably used as canbe stimulated in the ultra-violet, visible and/or infrared lightwavelength range and that emit in the ultra-violet, visible and/orinfrared light wavelength range.

In the case of one preferred embodiment of the invention, labelledregions of the biological object with a known distance from each otherare used as calibration targets. This labelling can be carried out withsuitable biochemical probes, for example.

The use of biological calibration targets has the practical advantage incomparison to the use of synthetic calibration targets, such ascalibration beads, that, along with the optical marginal conditions ofthe object, additional preparatively conditioned marginal effects in thecalibration are perceived during the calibration, such as for examplethe behaviour of an actual fluorescence signal against an unspecifiedbackground (which is determined by automatic image analysis algorithms).

Particularly suitable non-biological or synthetic calibration targetsare micro-beads with the same or a higher multispectral signature as orthan the measurement structures to be localised. These are treated inthe same way as the biological objects. Such calibration targets arepreferably fixed to object carriers in defined spatial arrays. They mayalready be fixed when the object carriers in question are manufactured,which is particularly advantageous for routine use.

In order to undertake the distance measurement according to theinvention using a microscope with axial tomography, the biologicalobjects are prepared with the measurement structures and the calibrationtarget(s) in or on a microcapillary or glass fibre as an object supportor object carrier. The capillary/fibre has a precisely defined diameter,of which a variety is possible. In order to affix this capillary/fibreto the microscope stage, the invention proposes a special mountconsisting of a rigid, preferably dorsiventrally flattened frame on oragainst which at least one bearing bush is mounted, in which amicrocapillary or glass fibre can be placed so that it can be rotatedaround its longitudinal axis and with its rotation axis perpendicular tothe microscope's optical axis. The bearing brush(es) should be set insuch a way that the rotation axis of the capillary/fibre runsperpendicular to the microscope's optical axis. The object of theinvestigation fixed or on the capillary/fibre is rotated by turning thecapillary/fibre directly, preferably using a torque motor. This has theadvantage that once the microscope lens has been focused and adjusted,it can be kept unchanged. Provision is made for a replaceable insert forthe frame to support or stabilise the capillary/fibre that has to berotated against sagging or displacement in the area of the frame recess.This insert can be made in particular of plastic or glass and have agrave-shaped excavation in the longitudinal direction of thecapillary/fibre. To facilitate handling, in particular during insertionin the frame, this insert can have one or more fixation slits.

The frame according to the invention preferably has the same externaldimensions as a conventional object slides, i.e. it is less than 77 mmlong and less than 27 mm wide, and it is very light, i.e. it weighs forexample only about 15 grammes. Using a computer controlled steppingmotor, the capillary/fibre can be turned through an exactly definedangle about its axis. As this rotation force is exerted directly on thecapillary/fibre and not on its mount, there is a considerable reductionin the danger that the capillary/fibre will be displaced and end upoutside the microscope's visual field or even that the whole microscopeplatform is disturbed or pushed.

In order to undertake the distance measurement method according to theinvention using a laser scan flow fluorometer, containing a fluid streamflowing freely or in an optical cuvette, and with one or more laser beamsource(s), whose laser beam(s) is/are focused on the central trajectoryof the fluid stream, the laser lens should be chosen and arrayed in sucha way that the laser beam(s) is/are focused in the form of a band on theobject trajectory (the central trajectory of the fluid beam leading theobjects).

For this purpose, the band form of this/these laser beam(s) can begenerated simply and preferably by interference fringes of two or moreintercrossing partial beams. The lenses and slits in the detection beampath are preferably selected and arrayed so that only the fluorescenceemission from the central interference fringe is depicted on thedetector. In addition, the detector should be divided into individualdetection pixels, in order to make a spatial resolution of thefluorescence emission in both the flow direction and perpendicular tothe flow direction possible. This has the advantage that the fluorescentmeasurement structures and/or calibration targets can be localised notonly in the flow direction, because of the movement of the object at aconstant speed, but also perpendicular to it.

In the case of a fluid stream in the z direction and a laser lens fromlaser beam(s) in the x direction and slit(s) and detector(s) in the ydirection, the method enables the resolution in two dimensions to beimproved. If a laser lens with laser beam(s) in the y direction andscan(s) and detector(s) in the x direction are added, there is animprovement in resolution in three dimensions. In both cases, it ispossible to generate point spread functions that are practicallyindependent of the object's orientation.

In accordance with on embodiment of the present invention, a distancemeasurement between gene sections of chromosomes in a cell nucleus(regardless of microscope type) is performed

In a cell nucleus, the chromatin of the individual chromosomes occupiesdefined partial regions. Within one or more of such chromosomal domains,the structures to be localised, i.e. the measurement structures, e.g.small chromosome sections such as genes or parts of genes, are labelledspecifically with a method, known to the state of the art, offluorescence in situ hybridising, using fluorochromes with certaindifferent spectral signatures M1, M2, M3, . . . The distances betweenthe labelled locations (the marked measurement structures) are below theclassical resolution, i.e. they are smaller than the half-intensitywidth of the maximum of the actual point spread function. The (object)structures (measurement structures) are marked in such a way that thespectral signatures are represented on the structures to be localised(measurement structures) with practically the same dynamic.

The biological object is prepared on a glass fibre with an exactlydefined diameter or in a round or quadrangular capillary with defineddimensions.

In order to determine the dimensions, microscopable preparations withcalibration targets are produced under the same physical and chemicalexperimental conditions as the object or the object structures to belocalised (=measurement structures).

The following are used as preparations or calibration targets, forexample:

a) micro-injectable spheres (beads) of one spectral signature(monochromatic):

According to known methods, the beads are each marked with onefluorochrome, i.e. monochromatically, and can be differentiated from thestructures to be measured (to be localised) in the object (themeasurement structures) on the basis of their size. Such calibrationbeads, which represent the spectral signatures of the measurementstructures present in the object, but otherwise are preferably identical(with regard to size, geometry, constituent material etc.) are injected.In other words, the spectral signatures of the measurement structuresand of the calibration targets are selected in such a way that, in thegiven experimental conditions, the fluorescence emissions originatingfrom them can be analysed separately. The monochromatic calibrationbeads are injected and fixed in such a way that the individual beadswith different spectral signatures are arrayed in clusters directly onthe surface of the glass fibre or the wall of the capillary, preferablyin a cross-section plane of the fibre or capillary. When precisionfibres and/or capillaries are used, the beads follow at given distancesfrom each other or from a plane, an axis or a line of reference.

b) micro-injectable test spheres (beads) with multispectral(polychromatic) signature and identical spectral dynamic:

According to the known method, the beads are each marked with all thespectral signatures occurring in the marked (object) structures(measurement structures). As a result, they can be injected in randompositions into the biological object (in this case a nucleus) to bemeasured. A nominal geometry as in a) is not necessary, as for eachsignature the chromatic barycentres should be localised in the sameposition. In order to differentiate between the marked (object)structures (measurement structures), the beads can either belong to adifferent class of size or also carry an extra spectral signature, whichis not found in most structures to be measured, i.e. the measurementstructures (according to the preparation protocol).

c) simultaneously labelled chromosome regions at a known distance of achromosome other than the one that carries the structures to belocalised (measurement structures):

The calibration target, i.e. in this case the chromosome regions at aknown distance from each other, are differently marked with the aid of atest combination of DNA sequences that bears the different spectralsignatures. The chromosomal calibration targets can be differentiatedfrom the (chromosome) structures to be localised (measurementstructures) for example by means of different fluorescence intensity orof a different intensity relationship between fluorochromes withdifferent spectral signatures or of the use of an additionalfluorochrome with a different spectral signature that was not used forthe fluorescent labelling of the measurement target.

It is also possible that the calibration target belongs to a differentclass of dimensions from the measurement structures to be localised.

The distance measurements according to the invention are carried outusing known far field microscopy equipment, consisting of a microscope,a photomultiplier and/or camera and a data processing unit. In this way,the distances between the calibration targets are measured usingfluorochrome markings with different spectral signatures. Thelocalisations thus measured (i.e. the target distances thus measured)are compared against the nominal localisations calculated using geometry(i.e. the real target distances), leading to the deduction of thespectrally caused shift. This shift is the calibration value for thedistance values measured between the (object) structures to be localised(measurement structures).

As this shift depends on the optical properties of the preparation (e.g.refractive indices in the nuclei and the preparation medium), thecalibration should take place in situ. In the case of the example inhand, this means that the calibration target should be located in thenucleus, alongside the (chromosomal) structures to be investigated andmarked (measurement structures). The distances between the (object)structures to be localised (measurement structures) or the distancesbetween the different colour signals or colour points of the measurementstructures in question, e.g. between the red fluorescent and the greenfluorescent colour point (from maximum intensity to maximum intensity orfrom barycentre to barycentre), are measured and this measurement valueis subjected to high precision correction by the shift detected with thecalibration target (caused by the different spectral signature).

In the case of calibration targets in the form of micro-injectable testspheres with multispectral (polychromatic) signatures, the chromaticshift is determined for each signature from the difference inlocalisation of the barycentres. The identification necessary for thispurpose of the fluorescence emission belonging to a calibration targetcan be undertaken for example by means of a volume-conservingthresholding process or by averaging the segmentation results in thecase of threshold variation.

The chromatic shift is determined in precisely the same way in the caseof calibration targets in the form of object regions with multispectral(polychrome) signatures marked with fluorochromes.

When centromer regions are hybridised with a test combination of suchDNA sequences that bind them all to the same chromosomal DNA sections,yet are labelled with fluorochromes with different spectral signatures,they are also particularly suitable as fluorochromatically markedcalibration regions. If the hybridisation is undertaken under even morestringent conditions, there are two labelling regions per cell nucleus;in the case of less stringent conditions, extra centromer regions arelabelled because of additional minor binding regions, so that the numberof the calibration regions increases. In some circumstances this is agreat advantage.

In accordance with a second embodiment of the invention, distancemeasurement between gene sections of a chromosome in a cell nucleususing an epifluorescence microscope with axial tomography

In order to undertake the method according to the invention, anepifluorescence microscope with axial tomography whose constructionprinciple is known is modified as follows:

Instead of an object slides, a mount according to the invention as inFIG. 1 for microcapillaries or glass fibres is placed on the microscopestage. This mount consists of a rigid, preferably dorsiventrallyflattened frame 1, on which a bearing bush 2 is mounted, in which amicrocapillary or glass fibre that can rotate about its own axis andwith its rotation axis perpendicular to the microscope's optical axis isset. The object of investigation fixed in or on the capillary/fibre isdirectly rotated by turning the capillary/fibre, either manually or bymeans of a torque motor. This has the advantage that once the microscopelens has been focused and adjusted, it can be kept unchanged.

In order to support or stabilise the capillary/fibre to be rotatedagainst sag and dislocation in the area of the recess 3 of the frame 1,a replaceable flat, slice-shaped insert 5 is set in the frame 1. Thisinsert 5 consists of plastic and has a grave-shaped excavation 8, whichstretches under and in the longitudinal direction of thecapillary/fibre. In the area of its border, the insert 5 is equippedwith two slit-shaped recesses 6, 7, which both stretch perpendicularlyto the edge of the border and facilitate the insertion of the insert 5in the frame 1. With a computer controlled stepping motor, thecapillary/fibre is rotated in the bearing bush through a known angleabout its own axis. The turning force is applied directly to thecapillary/fibre (and not to the mount), thus practically completelyavoiding the danger that the capillary/fibre is displaced and ends upoutside the microscope's visual field or even that the whole microscopestage is disturbed or pushed.

The biological micro-object, i.e. a cell nucleus, in which themeasurement structures to be localised are already marked withfluorochromes and that already also contains calibration targets (forpreparation see Embodiment 1), is located on the glass fibre or in themicrocapillary. The distance between two or more measurement structuresor calibration targets is smaller than the half-intensity width of theprinciple maximum of the axial actual point spread function. With theaxial tomograph, the object is rotated if necessary with automaticrefocusing. The rotation takes place in such a way that each distancebetween two measurement structures or calibration targets (i.e. betweentheir fluorescence intensity barycentres) is maximum. The maximumdistance measured is the equivalent of the actual distance.

If the only thing of interest is the distances between the measurementstructures or calibration targets, i.e. not their absolute spatialarray, it is now possible to proceed from one of the known measurementstructures or calibration targets to maximise and determine the distanceto a third measurement structure or a third calibration target. If thedistances between the measurement structures or the calibration targetsare larger than the half-intensity width of the maximum of the lateral(perpendicular to the optical axis) point spread function, then a singlespectral signature is sufficient; if on the other hand the distances aresmaller, the measurement structures or calibration targets must bedifferentiated by means of multispectral signature. The barycentres(maxima) of the signals are used for localisation. To the extent towhich the measurement structures investigated have a diameter that issmaller than the half-intensity width of the maximum of the actuallateral point spread function, all the refraction patterns of themeasurement structures or calibration targets will be determined bymeans of a sharp point spread function, so that the maxima can bedetermined optimally. If distances that are also greater than thehalf-intensity width of the maximum of the lateral point spread functionare permitted between the measurement structures or calibration targets,then it is only possible to determine all the distances between themeasurement structures or calibration targets for N ≧2 within a nucleuswith a single rotation if series of optical sections are recorded atevery rotation angle (see Embodiment 3 on confocal laser scanningmicroscopy).

If it is the absolute array of the measurement structures or calibrationtargets in space that is of interest, then the respective barycentresmust be determined accurately. The absolute localisation of themeasurement structures or calibration targets, i.e. the anglemeasurement, can be improved by multiple repetition of the wholemeasurement procedure and statistic evaluation.

In accordance with a third embodiment of the invention, a distancemeasurement between gene sections of chromosomes in a cell nucleus ispreformed a confocal laser scanning microscope.

A series of optical sections is taken from the biological micro-object,e.g. a cell nucleus, in which the measurement structure to be localisedhas already been marked with fluorochromes and that also alreadycontains calibration targets (for preparation see Embodiment 1). Themeasurement structures have l=1, 2 . . . L spectral signatures. Thespectral signature of the calibration targets are differentiated fromthose of the measurement volumes for example in terms of volume,diameter, intensity or of the number of spectral signatures (l=1, 2 . .. L+1). The images of the optical sections are taken separately for eachspectral signature and if necessary corrected according to thebackground.

For evaluation purposes, the calibration targets are first identifiedand the chromatic shift determined. To this end, the calibration targetsare localised under each spectral signature. In the case ofpolychromatic calibration targets, the spectral shift is the result ofthe difference between the localisations.

Secondly, the measurement structures are localised in parallel to or inconnection with the calibration. The position of the barycentres of theintensity signals measured is first determined in this way in eachspectral signature. The localisations are then corrected by the spectralshift resulting from the calibration measurements.

The corrected positions of the measurement structures are given inrelation to a point of reference. This point of reference may be arandomly chosen fix point in the object or the barycentre of acalibration target (e.g. a labelled chromosome region) or an otherwisechosen chromosome territory. It may however also be the barycentreco-ordinates of all the measurement structures within a chromosometerritory.

The measurement methods described for confocal laser scanning microscopycan also be undertaken in connection with axial tomography. In thiscase, the biological micro-object is rotated through a given angle and acomplete 3D image series is taken per angle, as described in Embodiment2. The size of the angle of rotation is determined in such a way thateach distance between two measurement structures is maximised. Theprocess is then undertaken for each 3D image series, as described inEmbodiment 2. The advantage of this rotation is that a distance is drawnbetween the localisation of two points that is determined in the lateralplane and thus on the basis of the sharpest (steepest) point spreadfunction.

Instead of undertaking the calibration and distance measurement betweenthe structures to be localised, i.e. the measurement structures, in thesame biological object as described above, it is also possible toundertake the calibration independently of the measurement structures onthe same type of biological objects. In this variation on the method,the difference between the fluorescence signals of the calibrationtargets and those of the measurement structures is facilitated. Usingthe values of the optical shift established with the calibrationtargets, it is possible to determine the calibration curves for thedistance measurements between measurement structures. This type ofcalibration curve makes statements for example about the spectral shiftas a function of the refraction index and absorption of the immersionmedium used, of the lens, filter and detection units used, of theevaluation algorithm used, of the biological object used, of the axialand lateral localisation of the measurement structures or calibrationtargets in them and so on. The use of information from this specialcalibration curve for distance measurements according to the inventionis particularly advantageous in cases in which a greater accuracytolerance may be used.

In accordance with a fourth embodiment of the invention, a distancemeasurement between gene sections of chromosomes in a cell nucleus isparfomed using a wave field microscope

The execution of the method according to the invention for measuringdistances using the wave field microscope is practically identical tothe method described in Embodiment 3. Unlike the confocal laser scanningmicroscope, the localisation is significantly more accurate in the axialdirection than the lateral direction in this case, as this is where thesharpest (steepest) point spread function is found. By combining thiswith axial tomography, the accuracy of the position determination anddistance measurement can be increased even further.

In accordance with a fifth embodiment of the invention, a distancemeasurement between gene sections of chromosomes in a cell nucleus usinga laser scanning flow fluorometer

The basic unit used for a laser scanning flow fluorometer is a flowcytometer, of the kind used routinely today for example for cellanalyses and sorting in immunology or haemotology. In this case, thebiological objects are arrayed in a fluid system in such a way that theyare conducted individually one after another through one or more laserfoci into the area of the central trajectory of a fluid beam that flowsfreely or in an optical cell. The biological object, in this example themetaphase chromosomes, are specifically labelled with one or morefluorescence markers and are excited selectively to fluoresce by thelaser beams. The fluorescence is normally registered in several opticaldetection channels separated by spectral filters and transmittedintegrally by photomultipliers into an amplified electric signal.

In order to be able to measure the fluorescence distribution withspatial resolution in one direction, so-called slit scan methods wereestablished. The biological object, in this case metaphase chromosomes,i.e. elongated objects measuring typically from 5 to 15 μm, areconducted by a fluid stream at a constant speed through one or morelaser beams that are focused so sharply in the flow direction that thefluorescence distribution is measured along the object in a timedependent way. On the basis of the known flow speed, it is possible totransform the mono-dimensional, time dependent fluorescence profiletaken per detection channel into localisation information. The flowspeeds are typically up to 10 m/sec and up to several thousand objectsper second can be analysed.

All slit-scan systems described before now in the literature have atypical focal width in the flow direction (measured from the actualpoint spread function) of at least about 2 μm (=resolution equivalent),as the focal depth decreases significantly with sharp laser focusing forfundamental reasons, so that a uniform resolution in the field ofpossible particle trajectories (typically 10 μm about the central axisof the fluid beam) can no longer be guaranteed. In addition, in the caseof the methods described in the state of the art, only amono-dimensional scan is possible.

In order to undertake the method of distance measurement according tothe invention in the area of nanometers, a laser scanning flowfluorometer whose principle of construction is known is modified by thefollowing arrangement (represented here by way of example for one laserand one detection channel):

Two coherent partial beams of the laser with identical intensity andeven wave fronts are focused on each other at a small angle in the fluidbeam and brought to interference in the focal area. The result is apattern of constructive and destructive interference, wherein theinterference fringes are orientated perpendicularly to the flowdirection. The half-intensity width of the interference fringe does notdepend on the width of the laser focus any more, but only on thewavelength and on the angle of incidence between the laser partialbeams. In the case of an angle of 28° between the two partial beams, thehalf-intensity width of the full interference fringe can be estimated atabout 500 nm for laser wavelengths of 500 nm.

The interference fringes can be generated with the depth necessary forall possible object trajectories of the fluid stream to be illuminatedwith the same fringe width.

As several interference fringes are generated at the same time, theobject information is ambiguous. The known method of convolving themeasured intensity distribution with the fringe pattern, i.e. with thepoint spread function, brings no or only a very small gain in resolutionwith respect to the conventional slit-scan process, as higherlocalisation frequency and thus resolution is lost because of a suitableselectable noise filter. According to the invention, the followingmethod is adopted instead of this:

Slits with a slit direction parallel to the orientation of theinterference fringe are built into the detection lens. Using suitablelenses, these slit-scans only take those fluorescences on thephotomultiplier that are stimulated between the first two minima of theinterference main fringe. In this embodiment, object trajectories of 6μm about the central fluid axis can be observed or investigated withpractically identical optical resolution using a 19° aperture angle ofthe detection lens (in air) and a minimal distance of ca. 1 μm.

In order to undertake more than just mono-dimensional scans, thedetection slit is divided into small rectangular elements-e.g. by meansof a CCD line. Signal amplifying optical elements can be pre-switched tothese CCD elements. The CCD elements are preferably arrayed in as largea segment of a circle as possible around the fluid stream. The detectionlens can then be partly replaced by microlenses in front of eachelement.

One alternative consists of arraying a laser lens of laser beam(s) inthe x direction and slit(s) and detector(s) in the y direction with afluid stream in the z direction, whereby an improvement in theresolution in two dimensions is achieved. If a further laser lens isarrayed with laser beam(s) in the y direction and slit(s) anddetector(s) in the x direction, the result is an improvement inresolution in three dimensions.

For each detection element, one fluorescence profile is taken throughthe object that is randomly oriented and moved at a constant speed inthe fluid stream through the central laser interference fringe. Smallfluorescent measurement structures or calibration targets in thebiological objects appear in the scan profiles as intensity peaks. Thefluorescent measurement structures or calibration targets can belocalised in the object on the basis of the intensity maxima and thearray of the scan profiles in question to the CCD elements. This canalso take place with identical spectral signatures, for example withregard to a defined object point of reference.

In the case of different spectral signatures, it is possible to use forexample laser beams with different wavelengths and/or several detectionunits. By means of calibration, the localisations of the individualmeasurement structures or calibration targets with different spectralsignatures can then be related to each other. The calibration andcomputation is then undertaken in terms of the above-describedembodiments for epifluorescence or confocal laser scanning microscopy.

In accordance with a sixth embodiment of the invention, using thedistance measurement method according to the invention is used foroptically controlling arrays of electronic components (electronic chips)

When electronic components (electronic chips) are arrayed, the plannedpresence of certain components and their geometric array must bechecked. As commercially available chip measurements are very small andthe individual components are closely related to each other, thenecessary control processes according to the state of the art areextremely expensive and not always satisfactorily accurate,

The distance measurement method according to the invention now enablesthe number and array of the components on a chip to be checked also incases when the minimal distance between individual chips is smaller oreven very much smaller than the half-intensity width of the principlemaximum of the point spread function.

For this purpose, the components are marked with fluorescent dyes, forexample on their surfaces, so that at least those components whosedistance is smaller than the half-intensity width of the principlemaximum of the point spread function are provided with fluorescent dyeswith different spectral signatures.

It is now possible to check the correct array of the components withoptical analyses, using the distance measurement methods according tothe invention and random far field microscopy methods, whereby insteadof the conventionally necessary lens with a relatively high numericalaperture, such as 1.3, it is now quite possible to use lenses withsignificantly smaller numerical apertures without for this reason losingany relevant information. With lenses with a smaller aperture, a largerworking distance between the lens and the object of investigation ispossible, which significantly facilitates handling and makes theinvestigations faster and more certain. Using the method according tothe invention, it is also possible the check the specified array ofthose components whose distance is smaller than 200 μm. This has theadvantage that it is possible to do without the use of technically verycostly optical near field methods and/or atomic force microscopy ortunnel electron microscopy methods.

Instead of electronic chips, it is equally possible to check DNA chipsor protein chips in a similar way.

What is claimed is:
 1. A method for applying far field microscopy and/orflow fluorometry to geometric distance measurements between objectstructures, wherein the distances can be smaller than the half-intensitywidth of the principle maximum of the point spread function, comprisingthe steps of: marking measurement structures to be investigated orlocalised with fluorescent dyes before, during or after preparation ofan object on or in an object carrier, wherein at least the measurementstructures to be localised whose distance is smaller than ahalf-intensity width of a principle maximum of a point spread functionare marked with fluorescent dyes with different spectral signatures,marking calibration targets with a defined size and array with the samefluorescent dyes, preparing the fluorescent calibration targets eithertogether with the measurement structures in or on an object carrier orseparately in or on an object carrier, investigating the measurementstructures and calibration targets under corresponding conditions,measuring two calibration targets with different spectral signatures toobtain actual measured values, dependent imaging and localisationbehaviour of the optical system used, the measurement determining adifference between the actual measured values and predetermined knownreal distance values to obtain a correction value using the correctionvalue to correct a shift brought about by the optical system in thedetection of different emission loci.
 2. The method of claim 1, whereinthe measurement structures and calibration targets are investigatedmicroscopically.
 3. The method of claim 1, wherein the measurementstructures and calibration targets are investigated at the same time. 4.The method according to claim 2, wherein the object is rotatedaxial-tomographically during the microscopic investigation.
 5. Themethod according to claim 4, wherein the object carrier comprises arotatable object support, and wherein the object is fixed in or on therotatable object support, the object support being made of a materialwhich is transparent for the light wavelengths used in theinvestigation, the material having a refraction index which differs atthe most by 12 percent from that of a surrounding medium, the materialhaving a cross section diameter which is less than or equal to 300 μm.6. The method according to claim 5, wherein the object support isrotated through anangle φ_(m),=360/3[∘],φ_(m),=360/4[∘] orφ_(m),=360/n[∘], wherein n is the number of planar sides of the objectsupport, and wherein a distance between the calibration targets and/orbetween measurement structures is measured for at least one spectralsignature at at least one of said angles.
 7. The method according toclaim 4, wherein the steps of measuring and determining comprisemarking, with a random spectral signature, one or more calibrationtargets B at a distance greater than the half-intensity width of theprinciple maximum of the actual point spread function from a barycentreof N measurement structures, measuring, via an automated imageprocessing method, the distances d_(ik)(i, k=1 . . . N, i≠k) of thebarycentres of the spectrally separated diffraction figures of the Nmeasurement structures and the distances d_(iB) between the Nmeasurement structures and the calibration target B; measuring, for onemeasurement structure, each of the stretches d_(ik) and d_(iB) in theplane of the narrowest point spread function and all other distances,whereby the object is rotated axial-tomographically each time through adefined angle φ_(m), correcting optical aberrations from the calibrationmeasurements and adapting a cosine function A_(ik)cos (φ_(m)+θ_(ik)) orA_(iB) cos (φ_(m)+θ_(iB)) with suitable phase dislocation to thecorrected measured distances d_(ik)(φ_(m)) and d_(iB) (φ_(m)), dividingmaxima A_(ik) and A_(iB) of the adaptation function of d_(ik) or d_(iB)by a multiplication factor and determining the maxima A_(ik) and A_(iB)as a Euclidean distance D_(ik) or D_(iB) between the N measurementstructures or between the measurement structures and the calibrationtarget B.
 8. The method according to claim 7, further comprisinganalogically evaluating a relative minima of a distance Z_(ik), z_(iB)in a plane orthogonal to a plane of the distances d_(ik), d_(iB), anddetermining a maxima as a function of the the relative minima of thedistance z_(ik), z_(iB) in the plane orthogonal to the plane of thedistance d_(ik), d_(iB).
 9. The method according to claim 7, furthercomprising using the 3D distances D_(ik) or D_(iB) in a system ofequations D² _(ik)=(x_(k)−x_(i))²+(y_(k)−y_(i))²+(z_(k)−z_(i))² D²_(iB)=(x_(B)−x_(i))²+(y_(B)−y_(i))²+(z_(B)−z_(i))² D²_(kb)=(x_(B)−x_(k))²+(y_(B)−y_(k))²+(z_(B)−z_(k))² to calculatepositions x_(i), y_(i), z_(i) and x_(k), y_(k), z_(k), as well asdistances x_(k)−x_(i), y_(k)−y_(i), z_(k−z) _(i) and x_(B)−x_(i),y_(B)−y_(i), z_(B−z) _(i).
 10. The method according to claim 7, furthercomprising determining co-ordinates and distances of the N measurementstructures as a function of the barycentres, wherein the barycentres aredetermined from barycentre centerings of the measurements to all pointsof reference.
 11. The method according to claim 7, further comprisingconvolving the positions received x_(i), y_(i), z_(i) and x_(B), y_(B),z_(B) with a point spread function with a half-intensity width, with theresolution equivalent achieved in each case and then representedgraphically.
 12. The method according claim 1 , comprising marking themeasurement structure(s) to be localised with fluorochromes withdifferent spectral signatures and the same dynamic.
 13. The methodaccording to claim 1, wherein marked regions at a known distance fromthe object are used as calibration targets.
 14. The method according toclaim 1, wherein micro-spheres with identical or higher multispectralsignatures than the measurement structures to be localised are used asthe calibration targets.
 15. The method according to claim 14, whereinthe calibration targets are fixed on object supports in a definedspatial array.
 16. The method according to claim 1, wherein saidinvestigating step is performed using a laser scan flow fluorometer witha flow cytometer that includes a fluid stream flowing freely or in anoptical cuvette with one or more laser beam sources whose laser beam(s)is/are focused on the central trajectory of the fluid stream, and with adetection system of detector(s), lens(es) and slit(s), wherein the laserbeam(s) are focused in a ribbon like shape on the object trajectory. 17.The method according to claim 16, wherein the ribbon like shape of thelaser beam(s) is generated by two or more intercrossing partial beams.18. The method according to claim 17, wherein the lenses and slits inthe detection beam path are selected and arrayed in such a way that onlyfluorescence emissions from the central interference fringe are depictedon the detector(s).
 19. The method according to claims 16, wherein thedetector(s) are divided into individual detection pixels and aresuitable for spatial resolution of the fluorescence emission in both theflow direction and perpendicular to the flow direction.
 20. The methodaccording to claim 7, wherein the co-ordinates and distances of the Nmeasurement structures are deduced on the basis of the barycentres,which are determined from barycentre centerings of the measurements toall points of reference.
 21. A mount for an object support comprising arigid, flattened frame with substantially a same length, width andheight as conventional glass object slides according to DIN 58884, atleast one bearing bush mounted on or against said frame, wherein one ormore tubular or gutter-shaped object carriers can be placed in the frameso that said object carriers can be turned around their longitudinalaxis with their rotation axis perpendicular to a microscope's opticalaxis, whereby the bearing brush(es) are mounted such that the rotationaxis of the object carrier is perpendicular to the microscope's opticalaxis, and that at least one terminal section of the object carrier isaccessible as a point of attachment for a torque force for rotating theobject carrier.
 22. The mount according to claim 21, wherein provisionis made for a replaceable insert for the frame, which is suitable forpartially or completely covering the recess in the frame.
 23. The mountaccording to claim 22, wherein the replaceable insert is made of amaterial selected from the group consisting of plastic and glass. 24.The mount according to claim 22, wherein the insert has a grave-shapedexcavation in the longitudinal direction of the object carrier.
 25. Themount according to claim 21, wherein the object carrier is amicrocapillary.
 26. The mount according to claim 21, wherein the objectcarrier is glass fibre.